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丙酮富集-毛细管电泳-质谱分析 O-糖肽。

Analysis of O-Glycopeptides by Acetone Enrichment and Capillary Electrophoresis-Mass Spectrometry.

机构信息

Department of Chemical Engineering and Analytical Chemistry, University of Barcelona , 08028 Barcelona, Spain.

出版信息

J Proteome Res. 2017 Nov 3;16(11):4166-4176. doi: 10.1021/acs.jproteome.7b00524. Epub 2017 Oct 10.

Abstract

Acetone precipitation was evaluated as a rapid, simple, low-cost, and efficient method for the selective purification of O-glycopeptides from enzymatic digests of glycoproteins. Ovalbumin (OVA), human and bovine α-acid glycoprotein (hAGP and bAGP), human apolipoprotein C-III (APO-C3), and recombinant human erythropoietin (rhEPO) were used to obtain enzymatic digests with a broad and varied set of peptides, N-glycopeptides, and O-glycopeptides. After digestion and before capillary electrophoresis mass spectrometry (CE-MS) analysis, the amount of ice-cold acetone added to the digests was optimized to maximize recoveries of O-glycopeptides. Furthermore, the different behavior of peptides, N- and O-glycopeptides was explained by studying with multivariate data analysis methods the influence of several physicochemical parameters and properties related to their composition and structure. Principal component analysis (PCA) and, afterward, partial least-squares discriminant analysis (PLS-DA) were used to identify the most significant variables and their importance to differentiate between peptides, N-glycopeptides and O-glycopeptides, or within these classes. This information was useful to understand precipitation of these compounds after addition of acetone and for the selection of the optimal conditions for purification of specific O-glycopeptide biomarkers. Special attention was paid to O-glycopeptide glycoforms of rhEPO because of their applicability in biopharmaceutical quality control and doping analysis.

摘要

丙酮沉淀被评估为一种快速、简单、低成本且有效的方法,用于从糖蛋白的酶解物中选择性地纯化 O-糖肽。卵清蛋白(OVA)、人及牛α-酸性糖蛋白(hAGP 和 bAGP)、人载脂蛋白 C-III(APO-C3)和重组人促红细胞生成素(rhEPO)被用于获得具有广泛多样的肽、N-糖肽和 O-糖肽的酶解物。在消化后和毛细管电泳质谱分析(CE-MS)之前,优化了向酶解物中添加冰冷丙酮的量,以最大限度地回收 O-糖肽。此外,通过研究与组成和结构相关的几种物理化学参数和性质,使用多元数据分析方法解释了肽、N-和 O-糖肽的不同行为。主成分分析(PCA),随后是偏最小二乘判别分析(PLS-DA),用于确定最显著的变量及其对区分肽、N-糖肽和 O-糖肽或这些类别内的变量的重要性。这些信息有助于理解添加丙酮后这些化合物的沉淀情况,并选择用于纯化特定 O-糖肽生物标志物的最佳条件。特别关注 rhEPO 的 O-糖肽糖型,因为它们在生物制药质量控制和兴奋剂分析中具有适用性。

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